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Fluorescent Detection of a Partially Unfolded Conformation of Beta-Lactoglobulin Using Squaraine Dyes



Six benzothiazole squaraine dyes were studied as fluorescent probes for detection of thermally-induced conformation changes of amyloidogenic protein beta-lactoglobulin (BLG). Majority of dyes have shown a strong fluorescent response when complexed with native protein, and at temperature of protein transition to “molten globule” state (65°C) an additional increase of dyes – BLG complex emission intensity up to 4.8 times was observed. The study of step-by-step BLG unfolding shows that dyes maximum fluorescence intensity was reached at temperatures around 55–59°C, which can be associated with the affinity of the dyes to tightly packed protein R-state. Squaraine dye P-15 containing diethylamino substituent in squaric core and a short N-ethyl pendent groups displayed the highest sensitivity to temperature induced conformational changes of BLG. This dye is proposed for studies as fluorescent probe for structural changes of globular proteins.